This invention relates to mixtures of partial ethers of cyclodextrins that have the ability to crystallize and the use of these mixtures to solubilize and stabilize other compounds.
Cyclodextrins are a group of compounds consisting of, or derived from, alpha-, beta- and gamma-cyclodextrins, so called parent cyclodextrins. Alpha-, beta- and gamma-cyclodextrin are crystalline oligosaccharides consisting of six, seven or eight glucose residues, respectively, with residues connected by 1 to 4 alpha glycosidic bonds to form a macrocycle. Cyclodextrins have the ability to include other compounds into their macrocycles. No chemical bonds are formed in this process, called formation of inclusion complexes. Cyclodextrins function as hosts, while the included compounds are called guests. Inclusion complexes formed by cyclodextrins have been useful for solubilizing and stabilizing guests such as drugs, ingredients of cosmetic and personal care products, dyes, agricultural chemicals and inorganic nanoparticles.
To improve the ability to form inclusion complexes, parent cyclodextrins have to be converted to derivatives. Each glucose residue of a cyclodextrin has one primary (OH-6) and two secondary (OH-2 and OH-3) hydroxyls that can be substituted (for example, converted into ethers). The final product obtained by such reactions can be either chemically distinct cyclodextrin derivatives, or mixtures whose components are partially etherified cyclodextrins that differ in number and position of substituents.
Known mixtures of cyclodextrin derivatives exist in stable, noncrystalline (that is, amorphous) form. Partial methyl ethers of cyclodextrins synthesized under strongly basic conditions provide widely used amorphous mixtures (U.S. Pat. No. 5,710,268; World Intellectual Property Organization 94/02516; U.S. Pat. No. 4,746,734; German Patent 294,267). Compositions of matter containing partially methylated derivatives of cyclodextrins can be synthesized under conditions of very low basicity, (minimal effective basicity). (See U.S. Pat. No. 5,681,828, U.S. Pat. No. 5,935,941; U.S. Pat. No. 6,001,821; World Intellectual Property Organization 92/22630.) The components of the latter mixtures are relatively few in number with substitution of the secondary hydroxyls predominating. Nevertheless, the mixtures prepared were found to exist in an amorphous state. Amorphous mixtures of partial 2-hydroxypropyl ethers of cyclodextrins (U.S. Pat. No. 5,096,893; U.S. Pat. No. 4,870,060) belong to this class and are produced using strong to moderately strong basicities.
Many chemically individual (and crystalline) cyclodextrin derivatives are known (A. R. Khan, et al., Chemical Reviews, 98, 1977-1996, 1998; A. R. Hedges, Chemical Reviews, 98, 2035-2044, 1998). Those of significant use include maltosyl-beta-cyclodextrin, which is produced industrially by enzymatic reaction (U.S. Pat. No. 4,931,389), and heptakis(2,6-di-O-methyl)-beta-cyclodextrin produced by reaction in strongly basic medium in presence of barium salts (U.S. Pat. No. 4,542,211). From the multitude of research generated compounds made by methods not suited for scale up, several chemically individual and crystalline methyl and 2-hydroxypropyl ethers of beta-cyclodextrin are known (K. Takeo et al., Die Starke 28, 226-227, 1976; K. Takeo et al., Carbohydrate Research, 187, 203-221, 1989; C. T. Rao et al., Journal of Organic Chemistry, 56, 1327-1329, 1991; J. Jindrich et al., Carbohydrate Research, 266, 75-80, 1995). It was believed that the crystallinity of these particular compounds was dependent on their being present as separate distinct entities, not as mixtures. The possibility of the existence of a crystalline lattice, which can accommodate a number of cyclodextrin derivatives of the kind described herein was not considered.
Water-soluble cyclodextrin derivatives, after their preparation, have to be separated from water-soluble by-products, i.e., inorganic salts and small molecular weight organic compounds. This has been done by diffusion-based processes, by demineralization with ion exchange resins, or by precipitation with organic solvents (A. R. Hedges, Chemical Reviews, 98, 2035-2044, 1998). In diffusion-based processes, the aqueous solution of product and by-products is brought into contact with a semi-permeable membrane which may, depending on the method of manufacture, permit only molecules that are smaller than those of the product to diffuse through. The other side of the membrane is in contact with water, or at least is covered by water. The smaller molecules of the by-product flow through the membrane to the aqueous side, where their concentration is lower. Molecules of water, since they obey the same physical law, flow in the opposite direction. This water flow leads to undesirable dilution of the desired product and can be prevented, or even reversed, by an increase of pressure on the product/by-product side. In the dialysis process, as used in the laboratory, the pressure on the product/by-product side of the membrane is not large enough to prevent fully the flow of water across the membrane into the desired product. Sturdy instrumentation that permits pressure large enough to prevent that flow or even reverse it, is known as reverse osmosis instrumentation. Use of reverse osmosis instrumentation to remove specific by-products, but not calcium salts, from cyclodextrin derivatives was disclosed in U.S. Pat. No. 5,831,081.
The present invention provides means for making certain mixtures of cyclodextrin derivatives that can be separated into crystalline and noncrystalline (amorphous) components. The crystallization does not lead to separation of individual compounds. Rather, both the crystalline and noncrystalline components remain as mixtures of more than one compound. Crystalline and amorphous states are associated with different and distinct advantages. The present invention enables access to compositions having the advantages of both states from one reaction product. The data provided herein show that components of both crystalline and amorphous states may have similar numbers of substituents per cyclodextrin residue: it is the position of substituents that is critical for determining crystallization properties of particular cyclodextrin products.
The invention provides compositions of matter comprising mixtures of partial ethers of parent cyclodextrins containing a crystalline component and an amorphous component wherein the crystalline component is comprised of cyclodextrins wherein total ether substituents are less than two times the number of glucose residues. It is possible to obtain therefrom a crystalline component containing mixtures of partial ethers of cyclodextrins. These crystalline cyclodextrins can form new, useful inclusion complexes with active agents such as drugs, agricultural chemicals, reaction agents, cosmetics, dyes and catalysts.
During methylation of beta-cyclodextrin using calcium hydroxide (i.e., base of minimal effective basicity) and low temperature, it was observed that crystals started to separate from concentrated solutions of the product. The crystalline phase thus obtained contained, in a single crystal, a multitude of chemically individual compounds, which among other aspects, differed by the number of methyls per molecule. This set apart the present composition of matter from compositions of previous art, in which existence of the single crystalline phase was associated with chemical individuality. Using these crystals as a seed, crystallization of other mixtures of partial methyl ethers of beta-cyclodextrin made in conditions of minimal effective basicity was possible. Crystalline phases were eventually obtained, but with much lower yields, from the corresponding derivatives of alpha-and gamma-cyclodextrin and from similar mixtures of partial hydroxypropyl ethers of beta-cyclodextrin, partial carboxymethyl ethers of beta-cyclodextrin and partial ethyl ethers of beta-cyclodextrin. In all of these cases, including partial methyl ethers of beta-cyclodextrin, reaction products contained both crystalline and amorphous phases.
Crystalline mixtures of partial methyl ethers of cyclodextrins of the present invention can be made in forms which vary in extent of overall substitution, in ease of crystallization, in solubility in water and in capacity to act as hosts in formation of inclusion complexes. Noncrystalline components of the compositions of matter of the present invention can also vary in degree of substitution and in their capacity to act as hosts.
The majority of cyclodextrin derivatives in use at present are amorphous mixtures. From the user""s point of view, the main advantage of amorphous character of a mixture is that it confers to its inclusion complexes high solubility. The principal disadvantage of the amorphous state of a mixture is that untoward properties of the cyclodextrin components are, given time, fully manifested. Components of amorphous mixtures of partial methyl and hydroxypropyl ethers of cyclodextrins, which have a high degree of substitution, are hygroscopic. This invariably leads to stickiness of the whole mixture and to difficulties in storage and mechanical processing. Another disadvantage is that the word xe2x80x9ccrystallinexe2x80x9d nearly universally conveys feeling of the purity; the word xe2x80x9camorphousxe2x80x9d conveys the very opposite.
From the view of producer, the amorphous state requires that the crude reaction product must fulfill all the purity criteria required in final products. None of the purification methods for use on amorphous compositions are sufficiently cost effective for large-scale production. Crystalline materials, on the other hand, can be inexpensively recrystallized until the desired purity is achieved. The effectiveness of recrystallization is well recognized by regulatory agencies. (Manufacture of pharmaceutical grades of glucose and sucrose serve as examples.)
In Example 1, a procedure is described for making the crystalline partial methyl ethers of cyclodextrin using calcium hydroxide. Results show that the level of methylation of the crystalline components of the mixture do not differ greatly in number of methyl groups per molecule from those of amorphous components left in mother liquors. The ease and rapidity with which crystallization of the purified multicomponent mixture of partial methyl ethers of beta-cyclodextrin occurs suggests that its components are isomorphous. In Example 1, it also is shown that reverse osmosis instrumentation can be used effectively to isolate the product.
In Example 2, it is shown that the crystalline phase can be isolated from many reaction products of cyclodextrins. The products of methylation of beta-cyclodextrin made using mild bases in addition to calcium hydroxide may also be used in compositions to provide crystalline products, albeit with lower yields. Results in Example 2 also show that a number of additional factors impact yields of the crystalline component of the mixtures. Yields of the crystalline component are improved when lower reaction temperatures and less alkylation agent are used. These factors are expected to impact crystallization potential since they diminish the number and variety of individual chemical compounds present in the reaction product. Surprisingly, there was a difference between alkylating agents, dimethyl sulfate giving higher yields than methyl iodide. Yields of the crystalline component were increased when barium ions were added to the reaction mixture. Partial methyl ethers of beta-cyclodextrin were found to crystallize much better than those of alpha- and gamma-cyclodextrins. Changes in size of substituent (e.g., moving from the smaller methyl to the larger ethyl, carboxymethyl, 2-hydroxypropyl) substituents also decreased the ability of derivatives to form crystals. When 2-hydroxypropyl groups were tested, only components with two or fewer hydroxypropyl groups per cyclodextrin residue were detectable in crystals obtained.
Results in Example 3 document the structure of components of the crystalline phase and described some of its properties. A single crystal of a methylation product of beta-cyclodextrin was grown large enough for all required analyses. That crystal contained 17% of water of crystallization. Analysis of the substitution pattern (J. Reuben, Carbohydrate Research, 1986, 157, 201-213 and references therein) of this crystal revealed that the only methylation which occurred was that on OH-2 hydroxyls and not all of these were methylated. Mass spectrum of the same crystal documented that species from one to seven methyl groups per molecule were present. These results prove that the crystal is formed by isomorphous 2-O-oligomethyl beta-cyclodextrins. The absence of methylation on OH-6 hydroxyls also was documented chemically as shown by ease of formation of tertiary butyl ethers. Average number of methyls per molecule of the crystalline product can be manipulated by changes in amount of methylation agent used in preparation. Increase in that number is accompanied by an increase in solubility in water. When amounts of methylation agent were increased substantially, species with more methyls than seven per molecule could be detected in crystalline phase by mass spectrometry. Results in Example 3 also show that the compositions of matter of the present invention are different from any partial methyl ethers of cyclodextrin of previous art.
In Example 4, it is shown that both the crystalline and amorphous components of partial methyl ethers of cyclodextrin preparations are good solubilizers. Hydrocortisone, a drug that has commonly been used as comparison standard in this field, was solubilized more efficiently using compositions of the invention than by currently available cyclodextrin compositions. Hydrocortisone was shown to increase the solubility of crystalline partial methyl ethers of beta-cyclodextrin in water. Example 4 also describes methods that can be used to produce liquid or solid pharmaceutical formulations. The same methods are expected to have applications for preparation of personal care products and formulations used in agriculture.
Results in Example 5 show that the crystalline mixtures containing partial methyl ethers of cyclodextrin are of low toxicity and that biological activity of guests is unaffected when using such cyclodextrin derivatives as hosts.